LISTERIA MONOCYTOGENES
PCR detection Kit
Product Number # MBK0001
50 REACTIONS
Listeria monocytogenes detection kit for use with PCR Thermal Cyclers.
FOR LABORATORY USE ONLY
NAME AND INTENDED USE
Identification of Listeria monocytogenes DNA by polymerase
chain reaction (PCR).
INTRODUCTION
Listeria monocytogenes is responsible of a serious food-borne
disease that affects especially newborn, pregnant women, the
elderly, and patients with impaired cell-mediated immunity (1).
Ingestion of contaminated food causes an infection, named
listeriosis, characterized by a variety of severe syndromes, such as
encephalitis, meningoencephalitis, septicemia and abortion (2).
Current bacterial identification methods are microbiological
culture isolation or serum agglutination test. The first procedure,
requiring a minimum of 5 days, is time expensive while the second
is not specific because of cross-reactivity between L.
monocytogenes and other Gram-positive bacteria. On the contrary
PCR-based detection systems are specific and sensitive and have
shortened analysis time (3). L. monocytogenes identification by
PCR methods has been successfully applied to pre-enriched
bacterial cultures (4), concentrated bacterial suspensions (5) or
extracted food components (6).
PRODUCT DESCRIPTION
The "LISTERIA MONOCYTOGENES PCR detection Kit" allows
the detection of L. monocytogenes DNA using Polymerase Chain
Reaction (PCR). PCR primers specifically detect all the L.
monocytogenes strains belonging to each of the three
evolutionary lineages defined by Jinneman and Hill (7) The kit
contains reagents and enzymes for the specific amplification of a
172 bp region of the L. monocytogenes genome. To identify
possible PCR inhibition, an internal control, giving an amplicon of
112 bp, is also supplied in the PCR mix.
KIT CONTENTS
L. monocytogenes Mix: 1000 µl (2 vials)
Hot-Rescue DNA Polymerase (5U/µl): 20 µl (100 U)
L. monocytogenes Positive control: 300 µl
Water: 1000 µl
OTHER SUPPLIES REQUIRED
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Disposable latex gloves.
Precision pipettes.
Sterile pipette tips.
Desktop microcentrifuge.
Thermal cycler.
Sterile PCR reaction tubes.
Sterile 1.5 ml tubes.
Sterile mineral oil (to prevent evaporation in thermal cyclers
without heated lid).
Agarose gel electrophoresis apparatus.
DNA size standard (100 - 1000 bp).
UV-Transilluminator.
STORAGE
Store the kit at -20°C.
Repeated thawing and freezing cycles may reduce the sensitivity
and should be avoided. It is suggested freezing the reagents in
aliquots for intermittently use.
GENERAL PRECAUTIONS FOR PCR
The operator should always pay attention to:
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use pipette tips with filter;
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store positive material (specimens, controls and amplicons)
separately from all other reagents and, if possible, add it to the
reaction mix in a facility separated space;
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do not use the same precision pipettes for reaction mix and
DNA-samples;
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thaw all components samples at room temperature before
starting an assay;
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when thawed, mix the components and centrifuge briefly;
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work on ice or in a cooling block.
PROTOCOL
DNA ISOLATION
Various protocols or Kits can be used to extract bacterial DNA from many food and clinical specimens or
bacterial enrichment cultures.
Carry out the DNA isolation according to the procedure making sure that the extracted DNA is free from
PCR inhibitors.
The protocols, tested by DIATHEVA, are available on-line in the “LIBRARY” section
For isolation of L. monocytogenes DNA from milk samples we recommend you to use the "LISTERIA
MONOCYTOGENES DNA isolation Kit: Milk" Product Number # MBK0002
PCR SET UP
The use of thin-walled PCR tubes for all amplification steps is recommended.
Include a positive control and at least one negative control (water) in each PCR run.
Before each use thaw all reagents completely, mix and centrifuge.
The PCR assay must be prepared following the pipetting scheme below:
FOR 1 REACTION
L. monocytogenes Mix
Hot-Rescue DNA Polymerase
Sample (*Preferably = 1µg DNA /reaction)
Total volume
39.8 µl
0.2 µl
10.0 µl
50.0 µl
*The sample can contain up to 1 µg L. monocytogenes DNA without inhibiting the amplification reaction
Make a master mix combining the reagents together in a 1.5 ml tube.
Aliquot 40 µl of master mix into each PCR reaction tube before adding 10 µl sample, negative and positive
control.
In order to avoid cross contamination, first pipet the negative control, then the samples and finally the
positive control.
Centrifuge briefly
For PCR thermal cycler without heated lid, each reaction mixture will have to be covered with a drop of
mineral oil.
THERMAL PROFILE
Program the PCR thermal cycler with the following parameters:
1 cycle
50 cycles
1 cycle
95°C for 15 min
95°C for 15 sec
61°C for 20 sec
72°C for 30 sec
72°C for 4 min
cool down to 4°C
AGAROSE GEL ELECTROPHORESIS
Mix 25 µl of the PCR reaction with 5 µl of DNA loading buffer. Separate the DNA in the presence of a
DNA standard, on a 2% agarose gel containing ethidium bromide, for about 45-60 min. The DNA standard
should be specific for the low range (100-1000 bp).
DATA ANALYSIS
The following results are possible:
Band pattern
Band at 112 bp
Band at 172 bp and possibly
an additional band at 112 bp
Interpretation
NEGATIVE SAMPLE
POSITIVE SAMPLE
NO DIAGNOSIS
(see troubleshooting section)
No band
Examples of positive and negative samples are showed in the following gel electrophoretic separation of
PCR products:
1
2
3
4
5
6
7
172 bp
112 bp
Lane 1:
Lane 2-5:
Lane 6-7:
ΦX174 DNA/BsuRI DNA molecular weight marker (MBI Fermentas)
Amplification of 50, 10, 4 and 2 Listeria monocytogenes DNA molecules.
Negative controls
TROUBLESHOOTING
No band of 172 bp from the positive control
No band of 172 bp (L. monocytogenes) and 112 bp
(Internal Control) from the sample PCR
Band of 172 bp from the negative control
Incorrect programming of the thermal cycler:
repeat the PCR with the correct settings.
Degraded reagent: store PCR reagents at -20°C and
keep on ice once thawed. Avoid multiple freezethaw cycles.
Incorrect tubes: use thin-walled PCR reaction
tubes.
PCR reaction inhibition: repurify the DNA sample
to remove inhibitors.
Contamination of PCR reaction: vigorous cleaning
is recommended before repeat the amplification.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
Schuchat A., Swaminathan B., and Broome C. V. (1991) Epidemiology of human listeriosis. Clin.
Microbiol. Rev. 4: 169-183.
Cooper J. and Walker R. D. (1998) Listeriosis. Vet. Clin. North. Am. Food. Anim. Pract.14: 113-125
Makino S. I., Okada Y., and Maruyama T. (1995) A new method for direct detection of Listeria
monocytogenes from foods by PCR. Appl. Environ. Microbiol. 61: 3745-3747.
Bansal N. S. (1996) Development of a polymerase chain reaction for the detection of Listeria
monocytogenes in foods. Lett. Appl. Microbiol. 22:353-356
Nogva H. K., Rudi K., Naterstad K., Holk A., and Lillehaud D. (2000) Application of 5'-nuclease PCR
for quantitative detection of Listeria monocytogenes in pure cultures, water, skin milk, and unpasteurized
whole milk. Appl. Environ. Microbiol. 66: 4266-4271.
Herman L. M. F., De Block J. H. G. E., and Moermans R. J. B. (1995) Direct detection of Listeria
monocytogenes in 25 milliliters of raw milk by a two-step PCR with nested primers. Appl. Environ.
Microbiol. 61: 817-819.
Jinneman K.C. and Hill W.E. (2001) Listeria monocytogenes lineage group classification by MAMA-PCR
of the listeriolysin gene. Curr. Microbiol. 43: 129–133.
Sambrook J., Fritsc, E.F., and Maniatis T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition.
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.